3.2.2. *LbrHSP100* Gene Replacement

sgRNA selection and replacement of the *LbrHSP100* gene were done following the same strategy. We obtained putative *LbrHSP100*-null mutants with sgRNAs set 3, targeting sequences in the N terminus of *LbrHSP100* ORF and set 4, targeting 5' and 3' non-coding sequences flanking the ORF for whole-gene deletion (Figure 3A). One cell population each emerged from the transfections and gave rise to multiple clones. Two *HSP100*–/– clones obtained with sgRNAs sets 3 and 4, respectively, were then selected for further genetic and phenotypic characterisation. NGS analysis indeed confirmed the target-specific disruption of the *LbrHSP100* ORF and the on-target integration of both drug resistance cassettes at the predicted genomic sites for both *HSP100*–/– mutants (Figure 3B; Figure S7). Western blot analysis using HSP100-specific antibodies [38] confirmed the lack of HSP100 in both mutants (Figure 3C).

**Figure 3.** CRISPR–Cas9-mediated disruption of the endogenous *HSP100* gene in *L. braziliensis.* (**A**) For targeting *LbrHSP100* (LbrM.29.1350), two sets of sgRNAs tested (set 3 and set 4) worked efficiently. sgRNAs set 3 (*LbrHSP100*-513-5'sgRNA and *LbrHSP100*-712-3'sgRNA) targeted disruption of the *LbrHSP100* ORF in the N terminus. sgRNAs set 4 targeted 5' and 3' non-coding flanking sequences for *LbrHSP100* whole-gene deletion. Two cloned *L. braziliensis HSP100*–/– lines were studied, *HSP100*–/– cl.1 and *HSP100*–/– cl.2, derived from transfection of set 3 or set 4 of *LbrHSP100*-targeting sgRNAs, respectively. (**B**) Whole genome sequencing of *HSP100*-null mutant lines. Sequence reads from each analysed strain were aligned to the reference DNA sequence consisting of chromosome 29 of *L. braziliensis* M2904 reference genome using Bowtie 2 software. The Y-axis represents the number of reads and the X-axis shows the nucleotide position (bp) on chromosome 29. Grey shaded areas denote complete lack of aligned reads. (**C**) Verification of *HSP100*-null mutants by Western blot analysis using anti-HSP100 (1/1000) antibody. Anti-HSP23 antibody (1/500) served as loading control. MW = Molecular weight in kilodalton.

To assess the fate of the Cas9/T7 construct (pTB007 episome) in the CRISPR-derived null mutants, we analysed Cas9 expression on the mRNA and protein levels by qRT-PCR andWestern blot, respectively. Cas9 protein was undetectable in the three *HSP23*–/– and two *HSP100*–/– mutant clones (Figure S8A,B), alleviating concerns over phenotypic, off-target Cas9 effects.
